La Discriminación en la Capital

GD is defi ned as an antibody-mediated autoim- mune process that affects the thyroid gland and the extra-thyroid tissues in about 90 % of the cases. The production of Autoantibodies tar- geted against the TSH receptor (anti-TSHR) leads to excessive synthesis of thyroid hormones; however, the underlying infl ammation and remodeling mechanisms in the extra-thyroid tis- sues are still ambiguous. Family occurrence of autoimmune thyroid disorders are frequently identifi ed in GD patients; for instance, in indi- viduals with GD ophthalmopathy 36 % have a fi rst or second degree relative affected either with GD or another Autoimmune Thyroid Disease (AITD). While in monogenic diseases Monozygotic (MZ) twins show complete con- cordance, in complex inherited disorders the concordance is incomplete, but is comparatively higher in Dizygotic twins (DZ). Cohort studies in twins have identifi ed a concordance rate of 0.36 and 0.03 for MZ and DZ respectively; moreover, the analysis of these data shows that 79 % of the predisposition to develop GD is attributable to genetics, while the specifi c indi- vidual environmental factors that are not shared by the twins may account for the remaining 21 % [ 13 ]. The estimated concordance for GD may be different for the populations studied; for exam- ple, la concordance for American twins is 0.17– 0.02 in MZ and DZ siblings, respectively, suggesting a small contribution of genes for GD in the American population versus Europeans. Studies in families and twins have clearly shown that GD is not the result of a single gene defect, but rather that it has a complex inheritance pat- tern; consequently, the predisposition results from multiple genes with individual and very modest effects. Most of the loci identifi ed repre- sent a low risk of disease (between 1.2 and 1.5).

From the genetic perspective, the studies are aimed at identifying an association between two or more genetic polymorphisms and a characteris- tic genetic trait. Association studies differ from binding trials where the same allele (or alleles) are associated with that trait in a similar manner to the population as a whole; in contrast, binding studies allow for different alleles to be associated with that trait in different families. These associated studies may be done among unrelated patients (cases) and healthy controls to identify markers that signifi cantly differ between the two groups or in family groups usually comprised of a sick indi- vidual and his/her parents. The association analy- sis is a very sensitive method to identify weak genetic risk factors and is currently the preferred method to study diseases with this type of pattern. Association analyses are based on a comparison between a group of patients with a particular out- come and ethnical “matched” controls; if a statis- tically signifi cant difference in the frequency of a variable is observed between the cases and the controls, the conclusion is that such variable is associated with the disease or the outcome [ 14 – 16 ]. The most frequent type of genetic vari- ability in humans occurs at specifi c sites in the genome where individual nucleotide variations develop from one member of the population to another. These sites are called Single Nucleotide Polymorphisms (SNPs). Most SNPs exist as alter- nate alleles; i.e., A or G; in average, two randomly selected human genomes have approximately three million differences in individual nucleotides between them; in other words, one per one thou- sand base pairs. Generally speaking, the SNPs are single nucleotide mutations giving rise to changes in one amino acid in the protein – the frequency of the minor allele shall be more than 1 % of the population- [ 17 ]. The identifi cation of complex disease genes involves four phases: Phase 1:

Analysis of candidate genes; Phase 2: Genome- wide association studies; Phase 3: Extended

genome association studies; and Phase 4: Whole genome sequencing.

The implementation of these technologies has led to further developments in this area, identify- ing at least seven genes whose variants have been associated with AITD, including: HLA-DR gene,

immunoregulatory genes (CTLA-4, CD 40, PTPN-22 y CD 25) and thyroid-specifi c genes: Tg gene and TSHR gene [ 16 – 18 ].

HLA-DR Gene The available evidence suggests

that the HLA (6p21) region predisposes to AITD; the alleles within the HLA class II region (genes DRB1 and DQA1 are the ones with the strongest association to GD). An important association has also been identifi ed in the HLA Class I region, HLA-C and HLA-B. GD Caucasian patients have an increased prevalence of the DRB1* 03 DQA1* 05 and DQB1* 02 haplotype, though these are not always present in other ethnic groups. In fact, it has been found that the HLA- DPB1 *05:01 is the major gene that predisposes to GD among the Asian population, in a sub-population of Chinese patients with an OR = 2.3 and a popula- tion attributable risk of 48 %. Other susceptibil- ity variables with independent effects include the B *46:01, DRB1 *15:02 and 16:02, whilst DRB1 *12:02 and DQB1 *03:02 are protective. Arginine in the position 74 of the HLA-DRb1 (Deb-Arg74) has been recently identifi ed as the critical DR amino acid that causes GD suscepti- bility. Furthermore, the presence of the glutamine amino acid at the position 74 of the DRb1 chain is protective against GD [ 15 , 18 ].

CTLA-4 (Cytotoxic T Lymphocyte Antigen-4)

The CTLA-4 attenuates T-cell activity. Its struc- tural resemblance to CD28, both in terms of the chromosomal localization as in terms of the exon- intron organization, suggests that both genes share the same origin. CD28 is a 44 KD mem- brane glycoprotein with a 202 amino acid sequence, whose gene is localized in the long arm of chromosome 2. It is the principal co- stimulating molecule in the activation of T cells, where it plays a broad range of roles. One of the most important effects identifi ed is the dramatic increase in the production of IL-2, 4, 5 & 13, in addition to other cytokines such as TNFα-, inter alia. These cytokines act as growth factors, with an autocrine and paracrine action, in addition to reducing the T-cell response threshold and pre- venting apoptosis. CTLA-4 is expressed in acti- vated T-cells, CD4 and CD8, at levels 10 and 100

times lower than those of CD28, but binds to CD80 and CD86 with a dissociation constant 20–50 times higher. Although a similar role was initially attributed to CD28 in the activation of T-cells, the most recent experimental fi ndings indicate that CTLA-4 has a negative regulatory action. Some trials have established that CTLA-4 is a GD-susceptible gene; in the Chinese popula- tion, for instance, the A49G and CT60 polymor- phisms are associated with increased susceptibility for GD. The Odds Ratio (OR) for A49G was 1.49, whilst for CT60 was 1.45 [ 18 , 19 ].

CD 40 Among the identifi ed susceptibility

genes in Graves’ Disease, CD40 is the only one involved with regulating the B cells response; the physiological ligand for CD40 is the CD154 (CD40L) molecule, that is expressed on the sur- face of the T helper cells. Binding of CD40 by CD40L helps to drive the resting B cell from G 0

into the cell cycle, playing an essential role in the activation and proliferation of B-lymphocytes. The CD40 gene has been associated with GD and a plausible explanation is the a C allele may induce an overexpression of the CD40 molecule, leading to the activation of B-lymphocytes with a prevalence of the Th2-type immune response. Bases on extended genome studies, CD40 is one of the candidates for GE. These studies have involved the 20q11 chromosome region – called GD-2 – as a susceptibility locus. The C allele in the rs 1883832 has been found to generate an OR = 1.6 for Graves’ Disease among Caucasians, though the same association has been identifi ed among the Japanese population [ 19 , 20 ].

The PTPN22 Gene (Protein Tyrosine

Phosphatase Nonreceptor-22) PTPN22 is

involved in the antigen adaptive response via the dephosphorylation and inactivation of the T-cells receptor. The PTPN22 gene has been associated with the presence of AITD and other autoim- mune diseases; it is localized at the 1p13 chromo- some and codes for the so-called signaling protein Lymphoid Tyrosine Phosphatase (PTP), which is a potent t-cell activation regulator. This protein inhibits T-cell activation through binding to signal transduction molecules such as Csk

kinase that mediates T-cell activation. The best association documented of the PTPN22 variants with autoimmune diseases, including GD is with rs2476601 (C1858T). This polymorphism has been studied and considered as a candidate sus- ceptibility gene for AITD in several ethnic groups (particularly Caucasians). The PTPN22 C1858T polymorphism triggers a change in the 620 posi- tion from an Arginine to Tryptophan, resulting in a less effi cient binding to Csk kinase. Hence, T-cells express T alleles that may be hypersensi- tive and lead to autoimmune disorders. However, the association of these polymorphisms with increased susceptibility in AITD has been vari- able, with both negative and positive associations due in part to the genetic heterogeneity among the populations studied and to other potential confounders [ 19 , 20 ].

Interleukin-2 Receptor α Gene (IL-2RA) This

gene codes for the α chain of the IL-2 receptor (IL-2R) complex, also known as CD25, which plays a key immunoregulatory role as an impor- tant auto-tolerance and immunity modulator. The IL-2RA gene has been associated with Graves’ disease [ 15 , 19 ].

Thyroglobulin (Tg) Tg represents one of the

major auto-antigens involved in AITD; however, the anti-Tg are not specifi c and occur in 80–90 % of the patients with Hashimoto’s disease and in 50–70 % of the patients with Graves’ disease (usually at low concentrations). The association of Tg polymorphisms with GD has been identi- fi ed with relapse of the disease following anti- thyroid treatment; nonetheless, these conclusions are based on studies with a small sample size that have not been homogenously replicated [ 14 , 18 ].

Fc Receptor-Like Gene (FCRL) This gene

codes for products that play a key role in the con- trol of B cell signals and it has been shown to be associated with autoimmune diseases. The FCRL3 is one of the fi ve genes preferentially expressed in B-lymphocytes and are structurally similar to the Fc receptors. The FCRL3 plays an inhibitory role in the B cells signaling process, resulting in B-cells disrupted tolerance and activity.

The association of SNP rs7528684 was repli- cated in Japanese subjects with GD and Systemic Lupus Erythematosus (SLE). The 1p21-23 cyto- band in which the FCRL resides has been identi- fi ed as a candidate locus for several autoimmune disorders with a strong association identifi ed for SNPs in that region, and increased susceptibility to GD in the Japanese population. This suggests that the origin of the association is a regulatory SNP in the FCRL3 promoter region. The rs7522061 SNP in the FCRL3 gene has also been associated with AITD in Caucasians, and more recently the SNP rs3761959 and the rs7522061 has also been associated with GD [ 13 , 19 , 20 ].

TSH Receptor (TSHR) TSHR and its ligand

-TSH- are critical thyroid activity regulators. The TSHR stimulating antibodies simulate the TSH action, resulting in the characteristic hyperthy- roid status. The TSHR is considered to exacer- bate the autoimmune process, as a key component for the onset of GD. The TSHR is made up of 10 exons that code for 764 amino acids of approxi- mately 95 kDa. The genetic variants of the TSHR probably stimulate autoimmunity in GD, particu- larly in individuals that exhibit other loci or gen- eral autoimmune risk; it is then possible for the TSHR genetic variants to infl uence the post- translational changes in the TSHR and/or the gene expression, thus increasing the risk for the TSHR to be an immune target; anti-TSHR can be found in 95–96 % of GD patients [ 15 , 18 ].

GD has a strong genetic component, and hence is infl uenced by family history. The inter- action between genetic susceptibility and envi- ronmental factors is extremely complex and poorly understood. Many of the genes associated with the onset of the disease are involved in other autoimmune diseases, including Type 1 Diabetes Mellitus, Rheumatoid Arthritis, and Multiple Sclerosis. This apparent genetic susceptibility convergence raises the risk for autoimmunity and when combined with specifi c risk factors triggers autoimmune diseases [ 21 ]. Several factors have been suggested to increase the risk of developing GD, including gender (the disease is more fre- quent in females than in males), prior infections

due to germs such as Yersinia enterocolítica , Yersinia pseudotuberculosis and Mycoplasma arthritidis, iter alia. The way in which these infectious agents may trigger a specifi c auto- antigenic response is controversial, though a large number of mechanisms may be argued. One of these mechanisms is the induction of an infl ammatory response leading to the production of pro-infl ammatory cytokines that may cause the aberrant over-expression of Class III MHC, resulting from the presentation of auto-peptides through the MHC molecules that lead to antigen- specifi c T-cell activation. The production of cyto- kines and their imbalance is caused by infection that may trigger the immune response. The post- partum period is a potential “predisposing” fac- tor, though clearly the pregnant woman regulates some genetic and autoimmune expressions that tend to activate after the end of the post-partum period. Consequently, while the likelihood of GD expressing during the post-partum period is higher as compared to gestation, the most plau- sible explanation may be the “attenuation” of autoimmune manifestations typical of pregnancy, rather than an increased risk during the post- partum period. Furthermore, during pregnancy fetal cells may reach the maternal circulation and may somehow infi ltrate various tissues – a pro- cess called “fetal microchimerism”. The infi ltra- tion of fetal cells into the maternal tissues infl uences the maternal immune response, though at a level that is diffi cult to quantify; these fetal

cells are valid candidates to explain the autoim- mune modulation of thyroid disease, both during pregnancy as during the post-partum period. The use of drugs such as amiodarone, lithium deriva- tives, α-interferon, anti-retrovirals, and monoclo- nal antibodies, increase the risk of developing GD. Smoking is another factor associated with the presence of GD and is considered a predictor of GD-related risk of hyperthyroidism. Most likely the existing relationship depends on the exposure time; active smokers have a higher risk than patients who smoked in the past or those that never smoked. These effect is also dose- dependent; i.e., the larger the number of cigarette packs smoked per year, the higher the frequency of Graves’ Disease, particularly in women that are heavy smokers [ 22 , 23 ].

In summary, the combination of genetic, envi- ronmental and endogenous factors is the etio- pathological foundation for GD (Fig. 5.1 ).